The role of PPARγ in TBBPA-mediated endocrine disrupting effects in human choriocarcinoma JEG-3 cells.

Honkisz E, Wójtowicz AK - Mol. Cell. Biochem. (2015)

Bottom Line:
Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level.Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control.These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells.

ABSTRACTThe goal of the present study was to investigate the action of TBBPA on PPARγ protein expression in vitro in human choriocarcinoma-derived placental JEG-3 cells. We also analyzed TBBPA for its action on placental secretion of progesterone and β-hCG, cell viability, and apoptosis. Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level. This alteration in PPARγ protein expression was accompanied by a decreased β-hCG level. Interestingly, co-treatment with the PPARγ antagonist GW9662 reversed the TBBPA-mediated changes in PPARγ protein expression but, according to β-hCG secretion, potentiated an inhibitory effect of TBBPA. Additionally, in our study, we assessed the ability of TBBPA to increase progesterone levels in JEG-3 cells compared with those of controls. Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control. The apoptotic action of TBBPA was also confirmed by Hoechst 33342 staining. These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells. Although co-treatment with antagonist of PPARγ reversed the TBBPA-mediated increase in this protein expression and restored it to the control level, it did not reverse the effect on β-hCG secretion. This indicated that the mechanism of TBBPA-induced changes in β-hCG secretion is PPARγ-independent.

Fig7: The upper panel shows a representative Western blot of PPARγ protein levels in JEG-3 cells treated with TBBPA (10 nM), GW1929 (10 µM), co-treated with TBBPA (10 nM) and GW1929 (10 µM), GW9662 (10 µM), co-treated with TBBPA (10 nM) and GW9662 (10 µM) for 48 h. The lower panel shows pooled data of three independent experiments. The Western blot membrane was cut into strips and probed with an anti-β-actin antibody to control the amounts for protein loading. PPARγ bands were quantified by densitometry. The results are shown as the percentage of PPARγ protein relative to the control. Data indicated with ***p < 0.001 reflects statistically significant differences relative to the control. Data indicated with ###p < 0.001 reflects statistically significant differences relative to TBBPA

Mentions:
The JEG-3 cell line that was used in this study was found to express the PPARγ protein, in agreement with previous studies [32]. Immunoblot analyses showed that compared with the control cells, TBBPA at concentrations of 10 nM and 10 µM increased the expression of the PPARγ protein in JEG-3 cells after 3–72 h of exposure (Fig. 6a, b). PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but remained above the control level. For a subsequent analysis, we measured PPARγ protein expression after TBBPA treatment at a concentration of 10 nM and a single time point of 48 h. To better understand the relationship between PPARγ protein expression and TBBPA treatment, we used a PPARγ agonist (GW1929) or antagonist (GW9662). Immunoblot analyses demonstrated that 10 nM TBBPA caused a 3.5-fold increase in the expression of the PPARγ protein compared with the control level (Fig. 7). Interestingly, treatment with agonist alone also caused a significant increase in PPARγ protein expression compared with that of the solvent control. Co-treatment with agonist or antagonist caused a modest but insignificant increase in the PPARγ protein expression.Fig. 6

Fig7: The upper panel shows a representative Western blot of PPARγ protein levels in JEG-3 cells treated with TBBPA (10 nM), GW1929 (10 µM), co-treated with TBBPA (10 nM) and GW1929 (10 µM), GW9662 (10 µM), co-treated with TBBPA (10 nM) and GW9662 (10 µM) for 48 h. The lower panel shows pooled data of three independent experiments. The Western blot membrane was cut into strips and probed with an anti-β-actin antibody to control the amounts for protein loading. PPARγ bands were quantified by densitometry. The results are shown as the percentage of PPARγ protein relative to the control. Data indicated with ***p < 0.001 reflects statistically significant differences relative to the control. Data indicated with ###p < 0.001 reflects statistically significant differences relative to TBBPA

Mentions:
The JEG-3 cell line that was used in this study was found to express the PPARγ protein, in agreement with previous studies [32]. Immunoblot analyses showed that compared with the control cells, TBBPA at concentrations of 10 nM and 10 µM increased the expression of the PPARγ protein in JEG-3 cells after 3–72 h of exposure (Fig. 6a, b). PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but remained above the control level. For a subsequent analysis, we measured PPARγ protein expression after TBBPA treatment at a concentration of 10 nM and a single time point of 48 h. To better understand the relationship between PPARγ protein expression and TBBPA treatment, we used a PPARγ agonist (GW1929) or antagonist (GW9662). Immunoblot analyses demonstrated that 10 nM TBBPA caused a 3.5-fold increase in the expression of the PPARγ protein compared with the control level (Fig. 7). Interestingly, treatment with agonist alone also caused a significant increase in PPARγ protein expression compared with that of the solvent control. Co-treatment with agonist or antagonist caused a modest but insignificant increase in the PPARγ protein expression.Fig. 6

Bottom Line:
Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level.Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control.These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells.

ABSTRACTThe goal of the present study was to investigate the action of TBBPA on PPARγ protein expression in vitro in human choriocarcinoma-derived placental JEG-3 cells. We also analyzed TBBPA for its action on placental secretion of progesterone and β-hCG, cell viability, and apoptosis. Our results showed that after TBBPA treatment at 10 nM and 10 µM, PPARγ protein expression increased in a time-dependent manner until 48 h and then slightly decreased at 72 h but was still above the control level. This alteration in PPARγ protein expression was accompanied by a decreased β-hCG level. Interestingly, co-treatment with the PPARγ antagonist GW9662 reversed the TBBPA-mediated changes in PPARγ protein expression but, according to β-hCG secretion, potentiated an inhibitory effect of TBBPA. Additionally, in our study, we assessed the ability of TBBPA to increase progesterone levels in JEG-3 cells compared with those of controls. Finally, in the present study, we demonstrated that TBBPA at all of the tested doses significantly increased caspase-3 activity compared with that of the vehicle control. The apoptotic action of TBBPA was also confirmed by Hoechst 33342 staining. These results showed the up-regulation of PPARγ protein expression after TBBPA exposure in human placental cells. Although co-treatment with antagonist of PPARγ reversed the TBBPA-mediated increase in this protein expression and restored it to the control level, it did not reverse the effect on β-hCG secretion. This indicated that the mechanism of TBBPA-induced changes in β-hCG secretion is PPARγ-independent.